The present invention relates to a method for the determination of follicle stimulating hormone (FSH) in aqueous solution, particularly body fluids. There are various reasons why it may be desirable to measure quantitatively and qualitatively FSH in a convenient and reliable manner. For example, it may be that the endocrine profile of a patient is needed for assessing the patient's pituitary functions. More commonly, in as much as FSH is necessary for follicle maturation and, therefore, fertility, the determination of a patient's circulating FSH may provide a useful clue to the "female" infertility problem.
The classical test for FSH is the Steelman-Pohley bioassay. It measures, for example, rat ovarian weight gain in response to administration of the hormone. Such bioassays are cumbersome, time consuming and expensive. Moreover, these assays lack precision, and sensitivity: there is a high variability between assays and the lower limit of detectability is in the .mu.g range compared to ng levels in, for example, serum. The current assay methods of choice are immunoassays and hormone-receptor binding assays. To perform an immunoassy, FSH must first be injected by suitable techniques into an appropriate test animal to elicit an immunological response and anti-FSH serum must be obtained. There are several common formats in which such an antiserum can be utilized for determining FSH. One commonly used format is competitive inhibition radioimmunoassay. A known amount of radiolabeled FSH, a suitable dilution of the anti-FSH antiserum and varying amounts of a test sample containing FSH are mixed and incubated. Any FSH present in the test sample will compete with the radiolabeled FSH for the available binding sites on the antibodies. If the antibody-antigen complexes are separated from the other portions of the mixture, the radiolabeled FSH recovered from the complexes will be diminished in proportion to the quantity of FSH present in the sample. Alternatively, the radioactivity in the other portions of the mixture will be increased proportionately and can be quantified. This and other formats are based on the specificity of antibody-antigen binding, i.e., on the antibodies' recognition of and affinity for antigenic determinants present in the FSH injected into the test animal. Even severely denatured FSH can be measured with the anti-FSH antiserum because some determinants remain recognizable. Consequently, this method measures both native (biologically active) FSH and denatured or modified (biologically inactive) FSH.
The hormone-receptor binding assay, on the other hand, is based on the specificity of hormone-receptor interaction in the normal physiological context. Thus, a metabolically defective hormone would elicit a positive reaction, i.e., reduce radiolabeled FSH binding, in an immunoassay but not in a receptor binding assay. The converse is true where a factor which inhibits hormone-receptor interaction is to be assayed. Thus, where an inhibiting factor is present, the receptor assay in the standard competitive inhibition format would give a positive reaction because the inhibitory factor, like FSH, will reduce the binding of radiolabeled FSH to the receptors. The inhibitory factor is a "FSH-like material" in this sense. The same inhibitory factor will neither inhibit radiolabeled FSH from binding to anti-FSH antibodies nor compete for the binding sites on those antibodies. Therefore, the inhibitory factor will give a negative reaction in an immunoassay. Generally speaking, the hormone-receptor binding assay is a more specific assay because, in most cases, specific receptor binding at the minimum requires the native conformation of a hormone in its biologically active form.
On a different level, the hormone-receptor binding and the immunoassay share fundamental similarities. Each rests on a ligand-receptor binding mechanism. Therefore, it is possible to utilize all the methodology previously developed for immunoassays within the context of the present invention. In particular, the previously developed signalling mechanisms are applicable. Notable examples are radioligands already referred to, enzyme-linked ligands (preferably linked to an enzyme indirectly through a noncovalent linkage) and ligands linked with a chemical label which may be fluorescent, phosphorescent or otherwise detectable. Accordingly, when the hormone-receptor binding procedure is coupled with a highly sensitive technique, for example, a radioassay, there is obtained a receptor binding radioassay method which possesses the degree of sensitivity of the radioimmunoassay techniques and, at the same time, the selectivity of bioassays. Furthermore, it is clear from the above discussion that a combination of a hormone-receptor binding assay and an immunoassay will provide more complete information than obtainable by each one alone.
Hormone-receptor binding assays have been developed for several hormones. For example, U.S. Pat. Nos. 4,016,250 and 4,094,963 disclose a receptor binding assay method and means for the determination of human chorionic gonadotropin (hCG), leutinizing hormone (hLH) and prolactin (PRL). European Patent Application No. 0108633 discloses improved methods and reagents for the determination of hCG and hLH. A hormone-receptor binding assay exists, albeit in less well developed form, for FSH. For example, Reichert and Bhalla, Endocrinology 94: 483-491 (1974), disclose a hormone-receptor assay for the determination of human FSH in human pituitary gland extracts. Briefly, this FSH assay comprises the steps of (a) contacting a sample suspected of containing FSH or FSH-like material with an agent capable of selectively binding the FSH or FSH-like material, for example, membranes containing FSH-receptors; (b) providing an entity for signalling whether the binding has taken place, for example, radiolabeled FSH; and (c) observing the signalling entity to determine the presence of FSH or FSH-like material in the sample, for example, by detecting the radiation emitted by the radiolabeled FSH which has become bound to the membrane receptors.
The disclosed Reichert and Bhalla method, however, does not work well with serum samples. Serum samples appear to contain small molecular weight interfering materials which interfere in the receptor assay and which can be removed by dialysis. See, e.g., Reichert and Leidenberger, Ovulation in the Human, Crosignani and Mishell (eds.), pp. 153-166, Academic Press, London (1976). Other investigations have found that merthiolate can be used to counter partially the interfering effects observed in receptor assays of serum. Minegishi, Igarashi and Waksbayashi, Endocrinol. Japan, 27(6): 717-725 (1980). The nature of these interfering effects is not clear. Serum may contain a hitherto unrecognized small ligand that binds receptor. It may contain a binding-inhibitory factor or a protease which cleaves off receptors, or some factor which non-specifically blocks the receptors. Notwithstanding all these uncertainties, serum remains the most conveniently obtainable clinical sample. Therefore, it is highly desirable to obtain a simple method and means which can overcome these problems in the determination of FSH, through means which do not require dialysis or other pre-treatment of the serum sample. Dialysis is time consuming, labor intensive and costly. Moreover, dialysis may cause the removal of small molecular weight materials which may be of clinical significance. Example 11 hereinbelow suggests that such materials do exist in serum samples of patients suffering from premature ovarian failure.
Furthermore, normal human serum samples contain about 4 to 25 mIU/ml of FSH. Turner, C. A. and Bagnara, J. T., General Endocrinology, W. B. Saynders & Co., Phila. (1976). Methods in the prior art can detect FSH levels in serum by RIA in excess of 3 mIU/ml but not at all by hormone-receptor binding assay. It is desirable to obtain an improved method and improved reagents for hormone-receptor binding assay that have greater detection sensitivity, and that may be used to assay serum samples.
Recently, Jia et al., J. Clin. Endocrinol. and Metabolism, 62: 1243-1249 (1986), disclosed a method applicable to serum samples. This method, however, has several serious drawbacks. It calls for preparations of explants of granulosa cells from immature rats and an in vitro culture assay that span a period of several days. Moreover, granulosa cells are not permanent cell lines and cannot be maintained indefinitely. Therefore, frequent fresh explants are necessary. Moreover, all serum test samples must be pretreated with polyethylene glycol and clarified by centrifugation to remove "non-specific" interfering materials. All these disadvantages add to the cost and complexity of such a test.